? Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Thaw GenSunBio DH5α competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by swirling or tapping the tube gently. Do not mix cells by pipetting.

? GenSunBio DH5α cells do not require IPTG to induce expression from the lac promoter. If blue/white screening is required to select for transformants, make sure that selective plates contain 50 μg/mL X-gal.

Transforming Competent Cells:

Perform the following before starting the transformation procedure:

? Equilibrate a water bath to 42℃.

? Warm the SOC. Medium or LB Medium to room temperature.

? Spread X-gal onto LB agar plates containing antibiotic, if desired.

? Warm the selective plates in a 37℃ incubator for 30 minutes (use 1 or 2 plates for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100μg/mL ampicillin.

Transformation Procedure:

Use this procedure to transform GenSunBio DH5α chemically competent E. coli. We recommend including the pUC19 control plasmid DNA in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.

1.Thaw, on ice, one vial of GenSunBio E. coli chemically competent E. coli for each transformation.

2.Add 1 to 5 μl of the DNA (10 pg to 100 ng) into a vial of GenSunBio cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1μl) of DNA into a separate vial of GenSunBio cells and mix gently.

3.Incubate the vial(s) on ice for 30 minutes.

4.Heat-shock the cells for 90 seconds at 42℃ without shaking.

5.Remove the vial(s) from the 42℃bath and place them on ice for 2 minutes.

6.Aseptically add 250μl of pre-warmed SOC or LB Medium to each vial.

7.Cap the vial(s) tightly and shake horizontally at 37℃ for 45 minutes at 150 rpm in a shaking incubator.

8.Spread 20-200 μl from each transformation on a pre-warmed selective plate and incubate overnight at 37℃. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (eg. remove 100 μl of the transformation mix and add to 900 μl of LB Medium) and plate 25-100 μl.

9.Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.

10.Invert the selective plate(s) and incubate at 37°C overnight.

11.Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Note:

?GenSunBio DH5α Chemically Competent E. coli should be stored at -80℃. Storage at -20℃ will result in a significant decrease in transformation efficiency. GenSunBio DH5α Chemically Competent E. coli lose efficiency whenever they are warmed above -80℃, even if they do not thaw.